Chemokines constitute a family of small cytokines that are produced in inflammation and regulate leukocyte recruitment (Baggiolini, M. et al., “Interleukin-8 and related chemotactic cytokines—CXC and CC chemokines,” Adv. Immunol. 55: 97–179 (1994); Springer, T. A., “Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration,” Annu. Rev. Physiol. 57: 827–872 (1995); and Schall, T. J. and K. B. Bacon, “Chemokines, leukocyte trafficking, and inflammation,” Curr. Opin. Immunol. 6: 865–873 (1994)). Chemokines are capable of selectively inducing chemotaxis of the formed elements of the blood (other than red blood cells), including leukocytes such as neutrophils, monocytes, macrophages, eosinophils, basophils, mast cells, and lymphocytes, such as T cells and B cells. In addition to stimulating chemotaxis, other changes can be selectively induced by chemokines in responsive cells, including changes in cell shape, transient rises in the concentration of intracellular free calcium ions ([Ca2+]i), granule exocytosis, integrin upregulation, formation of bioactive lipids (e.g., leukotrienes) and respiratory burst, associated with leukocyte activation. Thus, the chemokines are early triggers of the inflammatory response, causing inflammatory mediator release, chemotaxis and extravasation to sites of infection or inflammation.
Two subfamilies of chemokines, designated as CXC and CC chemokines, are distinguished by the arrangement of the first two of four conserved cysteine residues, which are either separated by one amino acid (as in CXC chemokines IL-8, γIP-10, Mig, PF4, ENA-78, GCP-2, GROα, GROβ, GROγ, NAP-2, NAP-4) or are adjacent residues (as in CC chemokines MIP-1α, MIP-1β, RANTES, MCP-1, MCP-2, MCP-3, I-309). Most CXC chemokines attract neutrophil leukocytes. For example, the CXC chemokines interleukin 8 (IL-8), platelet factor 4 (PF4), and neutrophil-activating peptide 2 (NAP-2) are potent chemoattractants and activators of neutrophils. The CXC chemokines designated Mig (monokine induced by gamma interferon) and IP-10 (γIP-10, interferon-gamma inducible 10 kDa protein) are particularly active in inducing chemotaxis of activated peripheral blood leukocytes. CC chemokines are generally less selective and can attract a variety of leukocyte cell types, including monocytes, eosinophils, basophils, T lymphocytes and natural killer cells. CC chemokines such as human monocyte chemotactic proteins 1–3 (MCP-1, MCP-2 and MCP-3), RANTES (Regulated on Activation, Normal T Expressed and Secreted), and the macrophage inflammatory proteins 1α and 1β (MIP-1α and MIP-1β) have been characterized as chemoattractants and activators of monocytes or lymphocytes, but do not appear to be chemoattractants for neutrophils.
Chemokines act through receptors which belong to a superfamily of seven transmembrane spanning G-protein coupled receptors (Murphy, P. M., “The molecular biology of leukocyte chemoattractant receptors,” Annu. Rev. Immunol., 12: 593–633 (1994); Gerard, C. and N. P. Gerard, “The pro-inflammatory seven transmembrane segment receptors of the leukocyte,” Curr. Opin. Immunol., 6: 140–145 (1994)). This family of G-protein coupled (serpentine) receptors comprises a large group of integral membrane proteins, containing seven transmembrane-spanning regions. The receptors are coupled to G proteins, which are heterotrimeric regulatory proteins capable of binding GTP and mediating signal transduction from coupled receptors, for example, by the production of intracellular mediators. Two of these receptors, the interleukin-8 (IL-8) receptors, IL-8R1 (interleukin-8 receptor type 1; Holmes, W. E. et al., “Structure and functional expression of a human interleukin-8 receptor,” Science, 253: 1278–1280 (1991)) and IL-8R2 (interleukin-8 receptor type 1; Murphy, P. M. and H. L. Tiffany, “Cloning of complementary DNA encoding a functional human interleukin-8 receptor,” Science, 253: 1280–1283 (1991)), are largely restricted to neutrophils and recognize the NH2-terminal Glu-Leu-Arg (ELR) motif, an essential binding epitope in those CXC chemokines that induce neutrophil chemotaxis (Clark-Lewis, I. et al., “Structure-activity relationships of interleukin-8 determined using chemically synthesized analogs. Critical role of NH2-terminal residues and evidence for uncoupling of neutrophil chemotaxis, exocytosis, and receptor binding activities,” J. Biol. Chem., 266: 23128–23134 (1991); Hébert, C. A. et al., “Scanning mutagenesis of interleukin-8 identifies a cluster of residues required for receptor binding,” J. Biol. Chem., 266: 18989–18994 (1991); and Clark-Lewis, I. et al., “Platelet factor 4 binds to interleukin 8 receptors and activates neutrophils when its N terminus is modified with Glu-Leu-Arg,” Proc. Natl. Acad. Sci. USA, 90: 3574–3577 (1993)). Five distinct CC chemokine receptors have been described, and are designated CC-CKR1, −2, −3, −4 and −5 (CC-CKR, CC chemokine receptor; Neote, K. et al., “Molecular cloning, functional expression, and signaling characteristics of a CC chemokine receptor,” Cell, 72: 415–425 (1993); Gao, J.-L. et al., “Structure and functional expression of the human macrophage inflammatory protein 1α/RANTES receptor,” J. Exp. Med., 177: 1421–1427 (1993); Charo, I. F. et al., “Molecular cloning and functional expression of two monocyte chemoattractant protein 1 receptors reveals alternative splicing of the carboxyl-terminal tails,” Proc. Natl. Acad. Sci. USA, 91: 2752–2756 (1994); Myers, S. J., et al., J. Biol. Chem., 270: 5786–5792 (1995); Combadiere, C. et al., Cloning and functional expression of a human eosinophil CC chemokine receptor,” J. Biol. Chem., 270 (27): 16491–16494 (1995); and Correction, J. Biol. Chem., 270: 30235 (1995); Ponath, P. D. et al., “Molecular cloning and characterization of a human eotaxin receptor expressed selectively on eosinophils,” J. Exp. Med., 183: 2437–2448 (1996); and Daugherty, B. L. et al., “Cloning, expression, and characterization of the human eosinophil eotaxin receptor,” J. Exp. Med., 183: 2349–2354 (1996); Power, C. A. et al., 1995, “Molecular cloning and functional expression of a novel CC chemokine receptor cDNA from a human basophilic cell line,” J. Biol. Chem., 270: 19495–19500 (1995); Hoogewerf, A. J. et al., “Molecular cloning of murine CC CKR-4 and high affinity binding of chemokines to murine and human CC CKR-4,” Biochem. Biophys. Res. Commun., 218: 337–343 (1996); Samson, M. et al., “Molecular cloning and functional expression of a new human CC-chemokine receptor gene,” Biochemistry, 35: 3362–3367 (1996)). The CC chemokine receptors occur on several types of leukocytes, including monocytes, granulocytes and lymphocytes, and recognize CC chemokines, but not CXC chemokines.
In contrast to monocytes and granulocytes, lymphocyte responses to chemokines are not well understood. Notably, none of the receptors of known specificity appear to be restricted to lymphocytes and the chemokines that recognize these receptors cannot, therefore, account for events such as the selective recruitment of T lymphocytes that is observed in T cell-mediated inflammatory conditions. Moreover, although a number of proteins with significant sequence similarity and similar tissue and leukocyte subpopulation distribution to known chemokine receptors have been identified and cloned, the ligands for these receptors remain undefined. Thus, these proteins are referred to as orphan receptors. The characterization of the ligand(s) of a receptor, is essential to an understanding of the interaction of chemokines with their target cells, the events stimulated by this interaction, including chemotaxis and cellular activation of leukocytes, and the development of therapies based upon modulation of receptor function.